PROJECT SUMMARY Chronic pain is a major unmet medical problem. The mechanisms that underlie the transition from acute (physiological) to chronic (pathological) pain are poorly understood and current therapies are inadequate. The proposal investigates colonic pain, with relevance to irritable bowel syndrome and inflammatory bowel disease. Proteases that are activated during injury and inflammation can signal to nociceptors by cleaving protease- activated receptor-2 (PAR2), which activates transient receptor potential channels and induces long-lasting hyperexcitability and nociception. Although proteases and PAR2 have been implicated in colonic pain, the signaling mechanisms that underlie persistent protease-induced pain are far from clear, and whether PAR2 is a therapeutic target for chronic pain is uncertain. The premise is that PAR2 is uniquely suited to transmit persistent nociception due to the irreversible mechanism of proteolytic (catalytic) activation, the capacity of the receptor to signal from endosomes, and the efficient mechanisms that mobilize intracellular receptor stores. Accordingly, antagonists of endosomal PAR2 and inhibitors of mobilization provide effective pain relief. These concepts will be examined in intact mice, isolated segments of mouse and human colon, and nociceptive neurons in culture. Approaches will include: behavioral assessment of nociception, electrophysiological analysis of nociceptor activation, and biophysical and imaging approaches to assess PAR2 trafficking and signaling in nociceptors. Mice expressing fluorescent PAR2 will be used to study PAR2 trafficking. Mice with targeted deletion of PAR2 on nociceptors, and unique lipid-conjugated antagonists of endosomal PAR2, will be used to determine whether PAR2 in endosomes of nociceptors is a therapeutic target for persistent colonic pain. The studies will provide insights into the mechanisms and treatment of chronic pain. They have implications for therapeutic targeting of G protein-coupled receptors; this 1,000-member family of receptors is the target of 40% of drugs. Since many activated receptors internalize and continue to signal, effective therapy requires endosomally-targeted drugs.